Method for mammalian cell transfection using 25-hydroxycholesterol

Abstract

The present invention relates to a process, a cell culture medium, and a kit for the rapid, high-efficiency transfection of mammalian cells with exogenous DNA. The process comprises incubating a cell culture in the presence of a transfection medium comprising a serum that is different from the serum in the normal growth medium used to grow the cells, and DNA to produce transfected cells. In a preferred embodiment the normal growth medium comprises fetal bovine serum and the transfection medium comprises a serum such as human, calf, horse, lamb, or pig serum. The transfection medium may further comprise an hydroxylated sterol such as 25-hydroxycholesterol.

What is claimed is: 
     
       1. A process for transfecting a mammalian cell with exogenous DNA comprising: growing a mammalian cell in a growth medium comprising fetal bovine serum;   replacing the growth medium comprising fetal bovine serum with a transfection medium comprising a second serum supplemented with 25-hydroxycholestrol, where the second serum is not fetal bovine serum;   adding a calcium phosphate-DNA precipitate solution to the transfection medium over the cell to form a calcium phosphate-DNA precipitate/medium mixture;   washing the cell; and   incubating the cell in the presence of a growth medium.   
     
     
       2. A process as recited in claim 1 wherein the fetal bovine serum is present at a concentration of 5% to 20%, by volume, of the growth medium. 
     
     
       3. A process as recited in claim 1 wherein the second serum is present at a concentration of 5% to 20%, by volume of the transfection medium. 
     
     
       4. A process as recited in claim 1 wherein the 25-hydroxycholesterol is present at a concentration of 0.1 μg/ml to 5 μg/ml. 
     
     
       5. A process as recited in claim 1 wherein the 25-hydrocholesterol sterol is present at a concentration of 1 μg/ml to 5 μg/ml. 
     
     
       6. A process as recited in claim 1 wherein the 25-hydroxycholesterol is present at a concentration of 2.5 μg/ml. 
     
     
       7. A process as recited in claim 1 wherein the calcium phosphate-DNA precipitate solution comprises: 125 mM calcium chloride;   25 mM N,N-bis(2-hydroxyethyl)-2-aminoethane-sulfonic acid, pH 6.9 to 7.0;   140 mM sodium chloride; and   0.75 mM sodium phosphate.   
     
     
       8. A process as recited in claim 1 wherein the cell is incubated in the presence of the calcium phosphate-DNA precipitate/medium mixture for 1 to 6 hours. 
     
     
       9. A process as recited in claim 1 wherein the cell is incubated in the presence of the calcium phosphate-DNA precipitate/medium mixture for 3 to 4 hours. 
     
     
       10. A process for transfecting a mammalian cell with exogenous DNA comprising: growing a mammalian cell culture in a medium supplemented with fetal bovine serum;   removing the medium and supplement from the cell;   adding a transfection medium supplemented with 10%, by volume, of a serum and 2.5 μg/ml 25-hydroxycholesterol to a mammalian cell culture, wherein the serum is selected from the group consisting of human, newborn calf, calf, horse, lamb, and pig sera;   adding a calcium phosphate-DNA precipitate solution to the transfection medium over the cell to form a calcium phosphate-DNA precipitate/medium mixture;   incubating the cell in the presence of the calcium phosphate-DNA precipitate/medium mixture at 35° C. to 37° C. in an atmosphere of 3% to 5%, by volume, carbon dioxide for less than 6 hours;   washing the cell; and   incubating the cell in the presence of medium supplemented with fetal bovine serum at 37° C. in an atmosphere of 5%, by volume, carbon dioxide.